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Shaomin Peng, Geliang Gan, Caihong Qiu, Lina Li, Ron A. Adelman, Lawrence J. Rizzolo; Comparison of Barrier Properties of RPE Derived from Two Human Embryonic Stem Cell Lines to the Properties of Human Fetal RPE. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5923.
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RPE-like cells can be derived from many human embryonic stem cell (hESC) and induced pleuripotent cell lines. Although these stem-cell derived cultures exhibit many RPE-specific properties, it is unknown whether their tight junctions display the same permeability and selectivity as native RPE, or whether all hESC-derived RPE are equivalent.
RPE isolated from 15-16 week human fetuses (hfRPE) was adapted serum-free medium (SFM). RPE derived from the H1 and H9 hESC lines was maintained in knockout serum replacement medium (KSR) or adapted to SFM. The barrier properties of RPE tight junctions were monitored using an Ussing Chamber with a voltage/current clamp. Global analysis of junctional proteins was performed by RNA-sequencing. The claudin family, which determines selectivity and permeability, was examined by quantitative RT-PCR (qPCR), immunoblotting and immunofluorescence.
Each culture condition yielded confluent, cobblestone monolayers of pigmented cells. For hESC-derived RPE, SFM increased and maintained a high TER relative to cultures in KSR medium. The TER was close to, but lower than, the TER of hfRPE that was adapted to SFM. The TER of H1-derived RPE tended to be higher than H-9 derived RPE. For Na+, K+, and Cl-, the selectivity for the hESC-derived RPE was similar to that estimated for hfRPE. In SFM, the expression of tight and adherens junction mRNAs was generally similar to hfRPE, but differences were found in the claudin family. By qPCR, SFM increased the expression of RPE-specific claudins in H1-derived RPE. The total claudin-19 levels were similar to native and cultured hfRPE. Similar results were obtained for H9-derived RPE, but the expression of several non-RPE claudins was also increased, and claudin-19a was overexpressed relative to claudin-19b. SFM also redistributed claudin-3 from the lateral membranes to the tight junctions.
Different preparations of hESC-derived RPE exhibit variable permeability properties, but SFM both decreases permeability and reduces variability. SFM induces the expression of RPE-specific claudins, but in H9-derived RPE can also induce non-RPE-specific claudins. This may explain why H1-derived RPE tends to have a higher TER than H9-derived RPE.
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